The overall aim of the proposed research and commercialization plan is to develop a technology that will yield a quantitative, multiplex IHC immunophenotyping assays for breast cancer clinical diagnosis and research. These assays will allow the oncologist to accrue the maximum amount of information on which to base diagnosis and treatment plans using a minimum amount of tissue, allow greater reproducibility of testing between clinical sites and provide quantifiable information within the context of tissue morphology. Validation of this platform technology with the breast cancer assays will allow us to expand use of the technology to other multiplex marker panels, allowing the possibility of a series of tests providing branched diagnosis resulting in better treatment decisions. This platform technology can then be expanded to other cancers and diseases encompassing both clinical diagnostics as well as research reagents. Expanding on our initial proof-of-principle studies, we will demonstrate multiplex fluorescent staining using our UltraPlex-BC1 panel (ER - estrogen receptor, PR - progesterone receptor, HER2 and Ki-67) on invasive breast cancer biopsies using our existing detection antibodies/technology whilst at the simultaneously develop second-generation detection reagents to expand our future plexing capacity. The UltraPlex-BC1 panel provides subtyping of breast biopsies into 4 major subtypes, one of which is a particularly aggressive form lacking the main ER/PR/HER2 markers, called triple negative breast cancer. Our second panel, Ultraplex-BC2 aims to subdivide the triple negative tumors into the more aggressive basal and less aggressive non-basal forms. Using our second-generation detection reagents we will demonstrate sensitive multiplex IHC staining of invasive breast tumors and compare this to current technology that employs single staining/enzyme labeled detection reagents. Finally we will validate both assays using a series of typed breast tumor tissues.